Wood–Ljungdahl pathway encoding anaerobes facilitate low-cost primary production in hypersaline sediments at Great Salt Lake, Utah

Abstract Little is known of primary production in dark hypersaline ecosystems despite the prevalence of such environments on Earth today and throughout its geologic history. Here, we generated and analyzed metagenome-assembled genomes (MAGs) organized as operational taxonomic units (OTUs) from three depth intervals along a 30-cm sediment core from the north arm of Great Salt Lake, Utah. The sediments and associated porewaters were saturated with NaCl, exhibited redox gradients with depth, and harbored nitrogen-depleted organic carbon. Metabolic predictions of MAGs representing 36 total OTUs recovered from the core indicated that communities transitioned from aerobic and heterotrophic at the surface to anaerobic and autotrophic at depth. Dark CO2 fixation was detected in sediments and the primary mode of autotrophy was predicted to be via the Wood–Ljungdahl pathway. This included novel hydrogenotrophic acetogens affiliated with the bacterial class Candidatus Bipolaricaulia. Minor populations were dependent on the Calvin cycle and the reverse tricarboxylic acid cycle, including in a novel Thermoplasmatota MAG. These results are interpreted to reflect the favorability of and selectability for populations that operate the lowest energy requiring CO2-fixation pathway known, the Wood–Ljungdahl pathway, in anoxic and hypersaline conditions that together impart a higher energy demand on cells.


Introduction
Autotrophs form the base of aquatic food webs and have a centr al r ole in ener gy flow to secondary consumers (Lindeman 1942 ).T hus , autotrophs and their activities influence the ov er all pr oductivity of aquatic ecosystems and their taxonomic and functional biodiversity (Smith 2007 ).In the photic zone of hypersaline lakes, the dominant primary producers are microbial, and include oxygenic photosynthetic organisms including both algae such as Dunaliella (Oren 2005 ) and Cyanobacteriota such as Euhalothece (Br oc k 1976 , Kanik et al. 2020 ) as well as anoxygenic photosynthetic bacteria such a members of the Chromatiaceae (Imhoff 2001 ).Far less is known of the primary producers in aphotic and benthic regions of hypersaline environments.Given the lo w er solubility of oxygen (O 2 ) in hypersaline w aters (Gar cia and Gor don 1992 ), a photic and benthic r egions of hypersaline environments are likely to be suboxic to anoxic.Together, with hypersalinity and associated low water activity (Grant 2004 ), low O 2 conditions would lead to pol yextr emophilic conditions (Capece et al. 2013, Merino et al. 2019 ) that could incr ease ener gy demands on cells (Hoehler 2007 , Shock andHolland 2007 ).
Hypersaline aquatic envir onments ar e typicall y formed thr ough e v a por ativ e pr ocesses (Gr ant 2004 ), and depending on their geological setting can vary widely in their ionic composition and in the availability of electron donors and acceptors (Oren 2013 ).Among the most widely available electron acceptors in anoxic zones of hypersaline aquatic envir onments ar e sulfate (SO 4 2 − ) and dissolved inorganic carbon [DIC; dissolved carbon dioxide (CO), carbonic acid (H 2 CO 3 ), bicarbonate (HCO 3 − ), and carbonate (CO 3 2 − ), r espectiv el y].The upper salinity limit for a variety of dissimilatory microbial processes, including those dependent on SO 4 2 − (i.e.SO 4 2 − reducers) and DIC (i.e.acetogens and methanogens) have been compiled based on laboratory studies of cultivars or via measurements of microbial activities associated with natural samples (Oren 1999(Oren , 2013 ) ).The salinity limit for dissimilatory SO 4 2 − reducers varies depending on whether they are complete or incomplete organic carbon oxidizers.Complete organic carbon oxidizers, or those that oxidize organic substrates (e.g.acetate) completely to CO 2 , are apparently restricted to < 12% salt (Brandt and Ingvorsen 1997 ).In contrast, incomplete oxidizers, or those that only partially oxidize organic carbon substrates , ha ve been identified at salinities up to 30% salt (Brandt et al. 2001 ), suggesting incomplete oxidizers can outcompete the complete oxidizers in high salt conditions.Autotrophic acetogens and methanogens that generate acetate and methane from H 2 and CO 2 , r espectiv el y, hav e upper salt tolerances of 24% and 11% (Zhilina et al. 1996, Ollivier et al. 1998 ), r espectiv el y.This suggests the possibility that acetogens can outcompete methanogens at ele v ated salt.These differences have been attributed to differences in the energy yields of those r espectiv e metabolisms and competition for the same electron donors/acceptors, namely H 2 and CO 2 (Or en 2013 ).As suc h, methanogens ar e thought to be more dependent on methylated compounds in hypersaline systems to minimize competition with acetogens for H 2 /CO 2 and to enable their coexistence (Kato et al. 2014 ).
Recent culti vation-inde pendent studies have identified putativ e SO 4 2 − r educers in the north arm of GSL, Utah (Dunham et al. 2020 ) and the nearb y shallo w subsurface halitesaturated sediments of the Bonneville Salt Flats (BSL), Utah (Mc-Gonigle et al. 2022a ).These organisms were closely related to the c hemolithotr ophic and hydr ogenotr ophic SO 4 2 − r educing gener a Desulfovermiculus (Beliak ov a et al. 2006 ) and Desulfonauticus (Mayilraj et al. 2009 ).Further, acetogens that were classified to the class Candidatus Bipolaricaulia (formerly referred to as candidate division Acetothermia or candidate division OP1; Hugenholtz et al. 1998, Hao et al. 2018 ) were identified in the BSL and GSL sediment columns .T he abundance of 16S rRNA gene transcripts affiliated with Ca.Bipolaricaulia was shown to increase with depth in the GSL sediment column (Dunham et al. 2020 ) and the r elativ e abundance of reads that mapped to a metagenome-assembled genome (MAG) affiliated with Ca.Bipolaricaulia was shown to increase with depth in the BSL sediments (McGonigle et al. 2022b ).Her e, we a ppl y meta genomic sequencing to thr ee depth interv als along a 30cm sediment core from the north arm of GSL to determine the composition, structure, and function of communities in anoxic hypersaline (r oughl y ∼30% NaCl) sediments.Specificall y, we seek to identify adaptations and putative trophic level interactions that enable habitation of these pol yextr eme envir onments.

Sample site description
GSL is a terminal lake in north-central Utah, USA (Hassibe and K ec k 1991 ).It exhibits a range of salinities due to localized freshwater input ( ∼95% of the total) to the southern end of the lake (Belovsky et al. 2011 ) (Fig. 1 ).The salinity of GSL was 20%-27% between 1900 and 1959 (Stephens 1990 ). Howe v er, in 1959, a r oc k and gr av el r ailr oad cause w ay w as constructed acr oss the lake, se v ering the lake into what is often r eferr ed to as a north arm (NA) and south arm (SA) (Cannon and Cannon 2002 ) (Fig. 1 ).The flow of water between the two arms is restricted to a recently constructed br eac h (Br own et al. 2023 ), whic h r esults in incr eased salinities in the NA ( > 30%) and decreased salinities in the SA (typically < 15%) (Baxter et al. 2005 ).

Sample collection and geochemical analyses
The sediment core from the NA of GSL was collected, processed, and analyzed as part of a prior study (Dunham et al. 2020 ).
Briefly, this sediment core was collected in Rozel Bay (41.43783 • , −112.67103 • ) on 9 May 2016 (Fig. 1 ).Two cores were taken ∼5 m from the shoreline and where the sediment water interface was ∼10 cm below the water air interface.Briefly, cores were taken by driving pol yvin yl c hloride pipes (6.3 cm diameter) into the sediment to a depth of 50 cm.One core was frozen upright with dry ice on site and r emained fr ozen during the transport to the lab, where it was transferred to a −80 • C freezer.This core was sectioned into 5 cm segments (pucks) in a −20 • C facility using a sterilized band saw.Core pucks were halved, with one half reserved for molecular (RNA) microbiological analysis and stored at −80 • C, while the other half was used for geochemical measurements, as described pr e viousl y (Dunham et al. 2020 ).A second core was collected and was stored upright on ice during transport back to the lab.This core was used for activity measurements .T he sediment cor e was c har acterized extensiv el y, with its miner ology, por e water c hemistry, and select micr obial activities measur ed and r eported pr e viousl y (Dunham et al. 2020 ).Pr e viousl y measur ed r ates of 14 C-bicarbonate assimilation, por e water dissolv ed CO 2 concentr ations, and por e water pH v alues in a parallel sediment core from the NA of GSL (Dunham et al. 2020 ) were used to normalize rates of 14 C-bicarbonate assimilation to rates of total DIC assimilation.Specifically, it was assumed that dissolved CO 2 (measured) and bicarbonate (not measur ed) wer e in equilibrium in the sediment cor e (pKa = 6.3).The Henderson-Hasselbach equation, along with the measured pH, were then used to calculate the concentration of bicarbonate.Concentrations of dissolved CO 2 and bicarbonate were then summed to estimate total DIC, which was then used to normalize rates of 14 C-bicarbonate to rates of total DIC fixed.Rates of 14 C-bicarbonate measured in disintegrations per minute (DPM) per gram dry-weight sediment (gdws) per hour were converted to μmols gdws −1 h −1 using the specific activity of the 14 C bicarbonate (52 μCi μmol −1 ).This was multiplied by the ratio of the amount of 14 C-labeled DIC added (1 μmol) to total DIC (from calculations above) to arrive at rates of total DIC assimilation.

DN A extr action and metagenomic sequencing, assembl y, and anal yses
Sediment cor e puc ks stor ed at −80 • C wer e thawed at r oom temper atur e in an ethanol-and UV-treated laminar flow hood.Based on variation in community 16S rRNA gene transcript sequencing, in particular the r elativ e abundance of transcripts associated with potentiall y nov el Ca.Bipolaricaulia (Dunham et al. 2020 ), ∼1 g subsections of sediment pucks from depths of 0, 5, and 30 cm were subjected to DNA extraction using the FastDNA Spin Kit for Soil (MP Biomedicals , Irvine , C A) following the manufacturer's instructions.Genomic DN A w as quantified fluor ometricall y (170-520 ng per sample) via the high sensitivity Qubit assay (Thermo Fisher Scientific, Waltham, MA).Shotgun metagenomic sequencing was conducted on genomic DNA from sediment core samples.Illumina libr ary pr epar ation and pair ed-end sequencing (2 × 150 bp) were conducted at the Josephine Bay Paul Center, located at Marine Biological Laboratory at Woods Hole Marine Biological Laboratory in Woods Hole, Massachusetts using the Illumina NextSeq platform.
Reads were trimmed and down-sampled with the TrimGalore v.0.6.0 and BBMap programs to cleave sequencing adapters and r emov e sequencing r edundancies as pr e viousl y described (Payne et al. 2019 ).Trimmed and down-sampled sequences were assembled individually and coassembled using Spades v.3.14.0 specifying default parameters .T he quality of the assemblies was then compared using v arious assembl y metrics and the metaquast pr ogr am (v.4.3) (Mikheenk o et al. 2018 ).Assembl y statistics ar e r eported in Supplementary Table S1 .The coassembled metagenomes resulted in substantially higher quality metagenome assemblies, and these were thus further used to c har acterize the comm unities.Assembl y statistics ar e r eported in Supplementary Table S1 .Assembled contigs were binned into MAGs using MetaBAT v. 0.26.3 (Kang et al. 2015 ) based on read depth and tetranucleotide frequency specifying the "verysensitive" setting.The assemblies were binned separately after coassembly and the quality, completeness, and level of contamination of each bin was assessed using Chec kM v. 1.0.5 (P arks et al. 2015 )."Outlier" contigs wer e r emov ed with RefineM v.0.0.23 (Parks et al. 2017 ).Only MAGs that exhibited > 50% estimated completeness and < 10% contamination (consistent with moderate to high quality genomes (Bo w ers et al. 2017 )) were retained for further analyses .T he MAGs were taxonomically classified using the GTDB-Tk v. 1.3.0 (Chaumeil et al. 2019 ) classifier and the bac120 and arc122 datasets for bacterial and archaeal classification, respectiv el y.Taxonomic designations are provided to the lo w est taxonomic rank that was formally recognized at the time of the writing of this manuscript.The r elativ e abundances of MAGs were estimated based on mapping of quality-filtered reads to those MAGs.Relative abundances are reported as the % of reads mapped to MA Gs.MA G contigs have been deposited in the National Center for Biotechnological Information (NCBI) Whole Genome Sequence database under bioproject accession PRJNA1036658.MAG taxonomy, estimated completeness, contamination, and abundance are reported in Supplementary Tables S2 and S3 .
A r epr esentativ e MAG fr om eac h OTU w as selected for do wnstr eam anal yses based on a set of empirically defined hier arc hical criteria.MAGs were selected firstly to maximize estimated genome completion, secondly to minimize estimated contamination, and, when necessary, thirdly to maximize their r elativ e abundance.MAG gene pr edictions wer e made using PROKKA v 1.11 (Seemann 2014 ) and r esultant pr otein annotated files were then uploaded to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Kanehisa and Goto 2000 ) using the KEGG Automatic Annotation Server (Moriya et al. 2007 ) to further examine the potential functionalities encoded by MAGs.
MAGs lacking homologs of these indicator proteins for autotrophic pathways were assigned as putatively heterotrophic.The carbon metabolism of cells was further assessed by examining MAGs for evidence of glycolytic , gluconeogenic , tricarboxylic acid, and pentose phosphate pathwa ys , based on KEGG outputs.Putativ e autotr ophic MAGs that also encoded gl ycol ytic , TC A, and pentose phosphate pathw ays w ere defined as facultative autotr ophs.Putativ e heter otr ophs that lac k ed terminal o xidases (described below) but that had components of gl ycol ytic pathways and fermentativ e ca pabilities (e.g.homologs of putativ e H 2 e volving [NiFe]-or [FeFe]-hydr ogenases) wer e classified as fermenters.While the a ppr oac hes used to classify an organism as autotrophic or heter otr ophic wer e conserv ativ e and used m ultiple lines of e vidence where possible, it is important to note that these MAGs are incomplete and it is possible that the lack of a given protein homolog(s) in a genome is attributable to this.

Phylogenetic analysis
To identify the most closely related organism with a genome sequence available for phylogenetic analyses, we subjected the beta subunit of RNA pol ymer ase (RpoB) or the alpha subunit of DNA gyr ase (GyrA) fr om a r epr esentativ e MAG (lo w est % contamination and highest % completeness) to BLASTp analysis against the NCBI nonr edundant pr otein database .T he MAG or genome with a corresponding homolog that was most closely related to the GSL MAG of interest was then used as the r efer ence genome to calculate whole-genome pair-wise amino acid identities (AAI).Pairwise AAI was calculated between the r efer ence and compar ator genomes using the Kostas Lab calculator ( http://en ve-omics .ce .gatech.edu/aai/ ).
To reconstruct the phylogenomic relationships of the Ca .Bipolaricaulota and Thermoplasmatota affiliated MAGs, the MarkerFinder pr ogr am (v.1.1)was employed to detect homologs of 40 universal single-copy housek ee ping phylogenetic mark er genes.In addition, publicly accessible Ca .Bipolaricaulota and Thermoplasmatota genomic data from the NCBI and the Genome Tree Database (GTDB) wer e incor por ated into the anal ysis .T he individual proteins encoded by the marker genes were aligned using Clustal Omega (v.1.2.4) (Sie v ers and Higgins 2018 ).Subsequentl y, the concatenated alignment was subjected to maximum likelihood (ML) phylogenetic analysis, employing IQ-TREE (v.1.6.11),with the optimal amino acid substitution model (LG + F + I + R4) among 168 potential models specified.The Bayesian information criterion as implemented in the model testing "TEST" function of IQ-TREE was used.To ensur e r obustness, ten independent phylogenetic anal ysis runs were executed and compared.The final ML reconstruction, yielding the most accurate representation, was selected as the definitive phylogenetic tree .T he support for the branches in the tree was evaluated by performing 1000 ultrafast bootstraps.

Sediment pore wa ter geochemistry, miner alogy, and microbial activities
Trends in the porewater geochemistry and minerology of the NA sediment column from GSL were described previously (Dunham et al. 2020 ) and select par ameters ar e r eintr oduced her e to pr ovide context for metagenomic data.Porewater total dissolved sulfide increased with depth from 6 μM at the sediment-water interface to 110 μM at a depth of 35 cm (Fig. 2 A) and por e water pH decreased in the N A betw een 0 and 30 cm in depth and then incr eased markedl y at 35 and 40 cm (Fig. 2 C).Por e water dissolv ed CH 4 concentr ations wer e uniforml y low along the depth tr ansect and ne v er exceeded 2 nM (Fig. 2 B) while por e water CO 2 gener all y decreased with depth (Fig. 2 D).The total organic carbon (C) to nitr ogen (N) r atio (C:N) in sediments fr om the NA cor e fluctuated between 24 and 32 throughout and did not follow an obvious pattern with depth (Fig. 2 E).Nonetheless, such high ratios in other lakes have been suggested to reflect input of terrestrial plant matter (Prahl et al. 1994 ) or could be indicative of substantial processing of algal or Cyanobacterial biomass (C:N ratio of ∼6-7; Redfield 1934 ), which tends to deplete the ratio.Porew aters w ere salt satur ated (pr ecipitated halite was detected thr oughout the column) and for simplicity their salinity was assumed to be the same as the waters ov erl ying the sediments ( > 26%) (data not shown).
Se v er al micr obial activities in sediment associated micr obial populations were also measured previously (Dunham et al. 2020 ) and ar e r er epr esented her e to pr ovide additional context for metagenomic data.This included DIC assimilation and acetate assimilation and miner alization.Importantl y, in the case of acetate assimilation/miner alization, these ar e pr esented as disintegrations per minute (DPM) gdws per hour since the amount of added radiolabeled substrate was k e pt constant but was not normalized to the concentration of native (unlabeled) substrates since these were not measured.Rates of DIC assimilation are presented as both DPM gdws −1 h −1 and as nmol gdws −1 h −1 following normalization to total DIC, as described below.Further, these activities wer e measur ed on a cor e that was not frozen on site (unlike that used for molecular and geochemical analyses) and as such, the sediments compacted during transport and do not perfectly align depth-wise with the molecular and geochemical data collected on the other column.Nonetheless, DIC assimilation attributable to microbial cells (primary production) was detected in communities associated with sediments throughout the column and did not v ary significantl y with depth (Fig. 2 G).Acetate assimilation and mineralization rates attributable to biology (secondary pr oduction) wer e also detected in comm unities associated with sediments (Fig. 2 H and I).Rates w ere lo w est at the sediment-water interface and increased with depth by se v er al orders of magnitude, with the highest rate of acetate mineralization detected at a Rates of DIC assimilation (measured in DPMs) based on assimilation of 14 C-bicarbonate were converted to rates of total DIC uptake by estimating the concentration of HCO 3 − from dissolved CO 2 and pH data (see the section "Materials and methods").
When converted, the rates of DIC assimilation ranged from ∼7 to 20 nmol C gdws −1 h −1 and these also did not follow a trend with depth.Despite the NaCl saturated nature of the sediment cor e, these r ates wer e within the range of those observed in other freshwater and marine sediments .For example , rates of dark DIC assimilation in four intertidal sediment cores collected from the Eastern Scheldt estuary, the Netherlands, ranged from 0.18 to 7.2 μmol C per cm 3 day −1 near the water-sediment interface only to drop to ∼ 0.02 μmol C per cm 3 day −1 or lo w er at depths of 2 cm or more (Boschker et al. 2014 ).Like wise, r ates of dark DIC assimilation in intertidal sediments from the German Wadden Sea ranged up to 0.1 μmol C per cm 3 day −1 near the water-sediment interface onl y to dr op to < 0.01 μmol DIC per cm 3 day −1 or lo w er at depths (Lenk et al. 2011 ).Assuming an av er a ge density of sand rich sediment of 2.0 g cm −3 (typical range of 1.7-2.3g cm −3 ; Manger 1963 ) and converting these to an hourly rate, they become 3.8-146 nmol C gdws −1 h −1 and 2.1-2.4 nmol C gdws −1 h −1 in the upper sediments from the Eastern Scheldt estuary and German Wadden Sea, r espectiv el y.In the lo w er sediments of these columns, rates drop to 0.06 ( < 2 cm depth Eastern Scheldt estuary core) and 0.41 nmol C gd ws −1 h −1 (10 cm de pth German Wad den Sea core).In other extr eme envir onments, suc h as hot spring sediments or proglacial sediments, rates of dark DIC uptake range from 10 to 100 nmol C gdws −1 h −1 and 0.  S1 .
r ange observ ed for other aquatic sediments and other extreme en vironments .

Taxonomic composition of the sediment core
The taxonomy (Fig. 3 ; Supplementary Tables S2 and S3 ), functional potential ( Supplementary Table S4 ), and % identity to organisms with available genomes ( Supplementary Table S5 ) of OTUs that comprised greater than 5% of the mapped reads within at least one depth were analyzed.Additional details of metabolic reconstructions of each OTU are presented in the Supplemental Information .At the 0 cm depth interval, OTUs were affiliated with the archaeal order Halobacteriales (OTUs 1, 3, 4, and 17), the bacterial family Salinibacteraceae (OTUs 15 and 12), the bacterial genus Thiohalorhabdus (OTU 2), and the bacterial class Ca.Bipolaricaulia (OTU 6) (Fig. 3 ; Supplementary Tables S2 , S3 , and S5 ).
The Halobacteriales affiliated OTUs were all predicted to be aerobic heter otr ophs based on genome reconstructions ( Supplementary Table S4 ).The presence of putative aerobic and heterotrophic Halobacteriales at 0 cm depth in the hypersaline GSL environment is unsurprising, as they have been detected and/or cultivated from GSL before (Post 1977, Baxter 2018, Kemp et al. 2018 )  MAGs corresponding to OTU 2 (Fig. 3 ; Supplementary Tables S2  and S3 ), which is closely affiliated with the bacterial genus Thiohalorhabdus ( Supplementary Table S5 ), encoded homologs of glycolytic pathwa ys , gluconeogenic , and TC A cycle proteins as well as homologs of phosphorubokinase and RuBisCO ( Supplementary Table S4 ), suggesting they correspond to facultative autotrophs.The presence of this OTU in surface sediments is consistent with decreased concentr ations of H 2 S near er to the surface of sediment column (Fig. 2 A).The genomic c har acterization of OTU 2 is also consistent with other c har acterization of this genus, whic h comprises halophilic, facultativ el y anaer obic autotr ophs that deriv e ener gy for carbon fixation primaril y thr ough sulfur oxidation (Sorokin et al. 2008 ).
The Ca. Bipolaricaulia affiliated OTU 6 ( Supplementary Table S5 ) is predicted to be anaerobic and to be a hydrogenotrophic acetogen (Fig. 4 ; Supplementary Table S4 ; discussed more below).The presence of an obligately anaerobic acetogen in a putatively oxy- Ho w e v er, it is suggested that the presence of abundant aerobic heter otr ophs in this depth interval may represent a strong enough sink for O 2 , facilitating the presence of this putative anaerobic acetogen.
At the 5 cm depth, the composition of the community did not c hange substantiall y r elativ e to the 0 cm depth, although the relative abundances of several OTUs changed (Fig. 3 ; Supplementary Tables S2 and S3 ).OTU 7, most closely affiliated with the bacterial order Desulfohalobiaceae ( Supplementary Table S5 ), was found at its highest in abundance at the 5 cm depth interval and is likely an O 2 -tolerant anaerobe (based on identification of Cyd homologs) and facultativ e autotr oph (based on detection of homologs of enzymes involved in the WL pathway; Supplementary Table S4 ), which is typical for other organisms of this order (Kue v er 2014 ).MAGs affiliated with OTU 7 encode a homolog of a group 1c [NiFe]hydr ogenase pr edicted to be involv ed in H 2 oxidation (Søndergaard et al. 2016 ) and homologs of Sat, Aps, and DsrAB.This suggests an ability to couple oxidation of H 2 to reduction of SO 4 2 − to gener ate ener gy for autotr ophy, whic h is consistent with c har ac-terizations of other members of this order (Mussmann et al. 2005, Meyer and Kue v er 2007, Santos et al. 2015 ).
Members of the order Halobacteriales (OTUs 1, 3, 4, and 17) gener all y decr eased in r elativ e abundance at the 5 cm depth interval (Fig. 3 ; Supplementary Tables S2 and S3 ).Similarly, the abundance of OTUs affiliated with the family Salinibacteraceae (OTUs 15 and 12) shar pl y decr eased at the 5 cm depth interv al and the abundance of Salinibacter (OTU 12) r epr esented less than 5% of comm unity.OTU 2, closel y affiliated with the genus Thiohalorhabdus ( Supplementary Table S5 ), also decreased in the 5 cm depth interv al.In contr ast, members of the class Ca.Bipolaricaulia (OTU 6) increased in abundance and a second OTU that is also closely affiliated with Ca.Bipolaricaulia (OTU 19) was detected in the 5 cm depth interval.OTU 19 encoded a suite of proteins that were similar to those encoded by OTU 6 and, as such, was also classified as an anaer obic hydr ogenotr ophic autotr oph, or mor e specificall y, an acetogen (Fig. 4 ; Supplementary Table S4 ; described in more detail below).The decreased abundance of aerobic heterotrophs and increased abundance of anaerobic autotrophs is consistent with an increase in the concentration of HS − with depth (Fig. 2 A).The decrease in aerobic heterotrophs is also consistent with the likely decrease of O 2 in the sediment column (Pace et al. 2016 ).T his , in turn, potentially facilitates the increase in anaerobic autotrophs.
Relative to the 0 and 5 cm depth, the composition of the comm unity shifted substantiall y in the 30 cm depth sediment interval (Fig. 3 ; Supplementary Tables S2 and S3 ).OTUs 1, 6, 18, 19, 25, and 29 were the only OTUs that comprised at least 5% of mapped reads in sediments at the 30 cm depth.Two of these OTUs were affiliated with the class Ca.Bipolaricaulia (OTUs 6 and 19), two were affiliated with the phylum Thermoplasmatota (OTUs 18 and 25), one was affiliated with the order Halobacteriales (OTU 1), and one with the Bacteroidetes (OTU 28) ( Supplementary Table S5 ).Of the OTUs present at the 30 cm depth, only OTUs 18, 19, 25, and 28 were not detected at ele v ated abundances ( > 5% of total ma pped r eads) at shallo w er depths in the sediment column.Notably, OTU 28 ( Bacteroides acidifaciens ) was the most abundant (26.9%) at the 30 cm depth column follo w ed b y OTU 6 (15.7%).Of the two OTUs affiliated with the phylum Thermoplasmatota , OTU 18 was identified as being part of the order PWKY01, an alphanumeric placeholder taxonomic designation (Rinke et al. 2021 ), and OTU 25 was identified as being a member of the Deep Hydrothermal Vent Eury ar chaeal Group 1 (DHVEG-1; Supplementary Table S5 ), which has r ecentl y been proposed to be renamed Thermoprofundales (Zhou et al. 2019 ).As described in more detail below, both OTUs 18 and 25 are predicted to be anaerobic and facultatively autotrophic.

Unique putati v ely autotrophic OTUs
OTUs not pr e viousl y described in GSL or that encode putativ e autotr ophic pathw ays w ere subjected to additional phylogenetic analysis to determine their relationships to other MAGs or genomes (Fig. 5 ).The metabolisms of these OTUs were further scrutinized to identify potential electron donors and acceptors that would provide energy to drive CO 2 fixation or that would inform on their ov er all mode of metabolism (if not putativ el y autotrophic) ( Supplementary Table S4 ).This included members of the Ca.Bipolaricaulia (Fig. 4 ) and Thermoplasmatota OTUs (described in text).

Candidatus Bipolaricaulia (OTUs 6 and 19)
A ML phylogeny was constructed to better depict the relationship of the two Ca.Bipolaricaulia -affiliated OTUs (GBDK taxonomic classification) from the NA sediment column in GSL to those identified pr e viousl y (Fig. 5 A).T he two Ca.Bipolaricaulia O TUs br anc hed basal to MAGs affiliated with the class Ca.Bipolaricaulia and its sister lineage UBA7950.The GSL OTUs formed a monophyletic lineage between existing members of the Ca.Bipolaricaulia and those that form the clade RBG16-55-12, that along with clade UBA1414 (not included in phylogen y), wer e formerl y known as "OPB41" (Hugenholtz et al. 1998 ).AAI scores indicate these OTUs are most closely related to single cell genomes corresponding to members of this clade (Merino et al. 2020 ).These cells, like many members of the Ca.Bipolaricaulia (Youssef et al. 2019, Colman et al. 2022 ), are acetogens that fix carbon via the WL pathway.Candidatus Bipolaricaulia MAGs pr e viousl y identified fr om halite crusts in the BSL (McGonigle et al. 2022a ), cluster within the Ca.Bipolaricaulia but are phylogenetically distinct from those identified in the NA of the GSL.
Phylogenetic placement of the GSL OTUs 6 and 19 between RBG-16-55-9 and Ca.Bipolaricaulales /UPA7950 lineages (Fig. 5 A), whose members often encode the WL pathway and that are described as acetogens (Youssef et al. 2019, Colman et al. 2022, McGonigle et al. 2022a ), suggests they may also be anaerobic autotr ophs and potentiall y acetogenic.Indeed, metabolic r econstruction of OTUs 6 and 19 r e v ealed homologs of all proteins involved in the WL pathway for CO 2 fixation (Fig. 4 ; Supplementary Table S4 ).These OTUs did not encode homologs of other terminal o xidases, suggesting the y are lik ely anaerobic autotrophs.Further, these OTUs encoded homologs of four [NiFe]-hydrogenase complexes classified in groups 1a, 3b, 3c, and 4 g (Søndergaard et al. 2016 ).Group 1a hydrogenase (HysAB) is positioned adjacent to the meth ylene-tetrah ydrofolate (MTHF) reductase subunits MetVF in the genome, suggesting it may be involved in providing electrons for MTHF reduction.The bidirectional group 3b hydrogenase, Hy-hBGSL, is predicted to allow for reversible NAD(P) + reduction with H 2 , while the group 3c hydrogenase MvhAGD is predicted to form a complex with HdrABC and bifurcate electrons from H 2 oxidation for sim ultaneous r eduction of ferr edoxin (Fd) and heter odisulfide (Kaster et al. 2011 ).The group 4 g hydrogenase, MahABCDGH, is colocalized on a contig with ion transporter and antiporter genes.T his complex ma y function in an ana pleur otic r ole by balancing the ratio of oxidized to reduced Fd and/or ion balance (Lie et al. 2012 ).Both OTUs also encode homologs of the Rnf complex, RnfABCDEG.Rnf links the Fd and NADH pools with the proton/sodium ion motive force.When the concentration of Fd is greater than N AD + , electron flo w is to NAD + and this is coupled to ion translocation out of the cell, conserving energy (Westphal et al. 2018 ).When the concentration of NADH is greater than Fd, Rnf works in r e v erse .T he Rnf complex plays an indispensable role in the energy metabolism of anaerobes as it maintains the ion gr adient acr oss the membr ane .T his gradient, in turn, allows for ATP synthesis via an F type ATP synthase .Importantly, GSL O TUs encode complete pentose phosphate and gl ycol ytic pathways as well as a nearly complete TC A cycle , suggesting the possibility that these organisms can possibly ferment as well.As such, these OTUs are designated as anaerobic facultative autotrophs that are likel y ca pable of hydr ogenotr ophic acetogenesis and fermentation.
Thermoplasmatota (OTUs 18,25,26,and 27) A ML phylogeny was constructed to better depict the relationship of the four Thermoplasmatota -affiliated OTUs from the NA sediment column in GSL to those identified pr e viousl y (Fig. 5 B).Refer ence genomes fr om the arc haeal E2 order (GTDB alphanumeric classification) and r ecentl y published genomes corresponding to the proposed archaeal orders Candidatus Haloplasmatales (Zhou et al. 2022 ) and Candidatus Thermoprofundales (Zhou et al. 2019 ) are also included.Two of the OTUs (OTUs 25 and 27) from GSL were taxonomically identified at the lo w est rank to the archaeal class E2 and order/family DHVEG-1 (GTDB classification).Phylogenetic analyses of these OTUs indicate that they br anc h within the clade comprising members of the E2 class, and mor e distinctl y, between two of the major r efer ence MAGs from the proposed Ca.Thermoprofundales order (MAGs M9B1D and M912D) (Zhou et al. 2022 ).These MAGs are referred to as Thermoprofundales for the remainder of this paper.
Phylogenetic reconstruction of OTUs 18 and 26 affiliated with the arc haeal famil y PWKY01 (GBDK classification) shows they both cluster within the proposed genus Candidatus Natronoplasma , one of four genera within the proposed order Ca.Haloplasmatales (Zhou et al. 2022 ).Consistent with this designation, both OTUs 18 and 26 share > 65% AAI with the proposed genomes that comprise Ca.Natronoplasma ( Supplementary Fig. S1 ), which is above the cutoff typically used to designate new genera.T herefore , it is likely that these two OTUs represent new species within this genus .T hese O TUs ar e r eferr ed to as Ca .Natronoplasma her ein.
The two GSL Thermoprofundales OTUs (OTUs 25 and 27) encoded nearl y complete gl ycol ytic and arc haeal pentose phosphate pathways ( Supplementary Table S4 ).These OTUs encoded homologs of [NiFe]-hydrogenases classified as groups 3b, 3c, and 4d (Søndergaard et al. 2016 ).The group 3b homolog is predicted to rev ersibl y couple H 2 oxidation to reduction of NADP + , the group 3c homolog is predicted to form a complex with Hdr and bifurcate electr ons fr om H 2 oxidation to the sim ultaneous r eduction of Fd and heterodisulfide, while the group 4d homolog is predicted to couple H 2 oxidation to Fd reduction accompanied by ion translocation.The only terminal oxidase identified in MAGs corresponding to these OTUs was a homolog of a putativ e fumar ate r eductase flavoprotein, indicating these OTUs ar e anaer obes.OTU 25 (but not OTU 27) encoded homologs of proteins involved in the r e v erse TC A cycle (rTC A), including ATP citr ate l yase (AclA), ATP citrate synthetase (CcsA), and citryl-CoA synthetase (CcsB).The MAGs corresponding to OTUs 25 and 27 have similar completeness at 87.9% and 85.7%, r espectiv el y, and the lack of rTCA homologs encoded in OTU 27 may allow cohabitation by minimizing nic he ov erla p.A pr e vious study of Thermoprofundales -affiliated MAGs r ecov er ed fr om brine pools in the Red Sea identified numerous homologs of [NiFe]-hydrogenase and a homolog of fumarate r eductase (Mwiric hia et al. 2016 ).Ho w e v er, unlike OTU 25, the Red Sea MAGs encoded the WL pathway.Neither OTU 25 nor OTU 27 encode a sufficient number of proteins that would suggest that they can fix carbon via the WL pathway.As such, OTU 25 is designated as an anaer obic, facultativ el y hydr ogenotr ophic autotr oph, which is consistent with previous studies of closely related members of the Thermoprofundales (Mwirichia et al. 2016 ), whereas OTU 27 is designated as a facultative anaerobic heterotroph.
OTUs 18 and 26 both encode nearly complete gl ycol ytic pathways ( Supplementary Table S4 ).Neither OTU encoded homologs of terminal oxidases, indicating they are anaerobic and likely fer-

Trends in metabolism as a function of depth
Each of the OTUs were assigned to functional guilds describing their potential to utilize O 2 (aer obe/aer otoler ant, anaer obe) and potential carbon metabolism (heter otr oph and autotr oph).The abundance of aerobes decreased, and the abundance of anaerobes increased with depth (Fig. 6 ), likely due to the reduced availability of O 2 as inferr ed fr om the incr easing concentr ations of sulfide.Similarly, the abundance of heterotrophs decreased with depth while the abundance of autotrophs increased with depth, perhaps due to decreasing availability of quality organic carbon.Interestingl y, the fr action of autotrophs that were anaerobic increased with depth, with nearly all the anaerobic OTUs inferred to be obligate anaerobes at the 30 cm depth interval.
Since anaerobes can utilize any of the six known pathways to fix CO 2 , the increased fraction of anaerobic autotrophs at depth prompted an analysis of the predominant carbon-fixation pathways in these or ganisms.Autotr ophs encoding the WL pathway were identified in each of the three depth intervals, and their abundance increased with depth.This was primarily attributed to the increased abundance of obligately anaerobic OTUs affiliated with the Ca.Bipolaricaulia and Desulfohalobiaceae as a function of depth.In contr ast, autotr ophs encoding the CBB c ycle w ere identified only in the 0 and 5 cm depth intervals and were affiliated with the genus Thiohalorhabdus .An autotroph encoding the rTCA c ycle w as identified at the 30 cm depth only and was affiliated with the Thermoprofundales .
Among the CO 2 -fixing pathways utilized by micr oor ganisms, the WL pathway is considered to be the simplest (fewest enzymes involved), most ancient (Russell and Martin 2004 ), and the least ener geticall y expensiv e (Bar-Ev en et al. 2010al. , Fuc hs 2011 ) ).In fact, the pathway is exergonic (Fuchs 1994 ) and releases enough free ener gy to driv e ATP synthesis if that ener gy is conserv ed (Thauer et al. 1977 ).T he WL pathwa y r equir es ∼1 ATP for the synthesis of 1 pyruvate from CO 2 whereas the CBB pathway requires 7 ATP, the rTCA r equir es 2-3 ATP, the DC/4-HP r equir es 5 ATP, and the 3-HP/4-HB r equir es 9 ATP.As suc h, anaer obic autotr ophs oper ating the WL pathway have been described as being given "a free lunch that they are paid to eat" (Shock et al. 1998 ).
T he WL pathwa y is the only autotrophic pathway that is exclusiv el y found in anaer obes, likel y due to the O 2 sensitivity of many of its enzymes (Ragsdale andPierce 2008 , Fuchs 2011 ).The utilization of this lo w er energy demanding pathway may be consistent with the lo w er energetic yields of anaerobic metabolisms (Thauer et al. 1977, Bar-Even et al. 2010 ).Based on the distribution of autotrophic pathways among cultivars that operate low energy yielding metabolisms (anaerobes) or in environments that impose high energy stress, it has been hypothesized that the WL pathway allows cells to fix CO 2 under conditions that would be otherwise unfav orable (Monto y a et al. 2012 ).The incr eased pr e v alence of the WL pathway with increasing depth in the NA GSL sediment column supports this hypothesis, in particular due to the high ener getic str ess that autotr ophs ar e likel y to experience due to pol yextr emophilic conditions (e.g.hypersalinity and euxinia) that would be incr easingl y encounter ed with depth.The identification of diverse and often novel WL pathway encoding anaerobic extremophiles in the euxinic sediments of GSL call for additional efforts to cultivate these organisms for detailed physiological and biochemical study and to further c har acterize the biodiv ersity of anaerobic sediments in hypersaline en vironments .Such efforts will help identify additional adaptations that allow life to thrive under pol yextr emophilic conditions on Earth and potentially on other planetary bodies.

Figure 1 .
Figure 1.Map of GSL with the location in the NA, where the sediment column was collected indicated by a black dot.The railroad causeway separating the NA from the SA and the primary rivers draining into GSL are indicated.Base map National Agriculture Imagery Program (NAIP) imagery sourced from United States Department of Agriculture (USDA) and river shapefile modified from the National Hydrologic Dataset (NHD).State shapefile within the inlay map is from the United States Census Bureau's Master Address File/Topologically Integrated Geogr a phic Encoding and Referencing (MAF/TIGER) Database (MTDB).

Figure 3 .
Figure 3. Taxonomic composition of MAGs r ecov er ed fr om depths 0, 5, and 30 cm in a sediment column from the NA of GSL.MAGs were compiled into OTUs using an ANI of > 95%.OTUs that were > 2% relative abundance of total reads mapped to MAGs are shown; all others are in the "other" category.The domain of each OTU is denoted as (A) for Archaea and (B) for Bacteria.OTUs were classified to the highest taxonomic rank using GTDB-Tk and this is indicated by abbr e viations: (c) class le v el classification, (f) family level classification, (g) genus level classification, and (s) species level classification.OTU designations are indicated in brackets and full taxonomies are reported in Supplemental TableS1.
Additionally, OTU 2 encoded homologs of a group 1a [NiFe]hydr ogenase pr edicted to be involv ed in H 2 oxidation (Søndergaard et al. 2016 ), Sr eABC involv ed in anaerobic elemental sulfur (S 0 ) reduction (Laska et al. 2003 ), SoxAB involved in thiosulfate (S 2 O 3 2 − ) oxidation (Wodara et al. 1997 ), Sqr involved in sulfide (HS − ) oxidation (Shahak and Hauska 2008 ), and Cox involved in O 2 r espir ation.T his is consistent with this O TU being capable of coupling oxidation of H 2 , HS − , or S 2 O 3 2 − to the reduction of O 2 or S 0 to provide energy for autotrophy.

Figure 5 .
Figure 5. Phylogenomic reconstruction of putative select OTUs in a sediment column collected from the NA of GSL.(A).Phylogeny of Ca.Bipolaricaulia affiliated OTUs 6 and 19 in relation to closely related taxonomic lineages.Genomes from the re presentati ve Thermotogota ( Thermotoga maritima MSB8, Fervidobacterium pennivorans DSM 9078, and Pseudothermotoga thermarum DSM 5069) were used as the outgroup.(B) Phylogeny of Thermoplasmatota affiliated OTUs 18, 25, 26, and 27 in relation to closely related taxonomic lineages.Genomes from the order Halobacteriales were used as the outgroup.Re presentati ve sequences (M9B1D and M912D) from archaeal E2 order (proposed Candidatus Thermoprofundales ) are bolded.Each branch of the tree displays bootstr a p support v alues (out of 1000 bootstr a ps).

Figure 6 .
Figure 6.Inferred metabolic potentials of MAGs recovered from 0, 5, and 30 cm depth intervals in a sediment column from the NA of GSL.(A) Carbon source and O 2 utilization were inferred by first clustering MAGs into OTUs based on 95% ANI and then functionally annotating a MAG re presentati ve of each OTU.Relative abundances for each OTU were determined by mapping reads back to each constituent MA G .OTUs encoding autotrophic pathw ays w er e classified as autotr ophs, while those lac king autotr ophic pathw ays and possessing pathw ays indicativ e of heter otr ophy (e.g.gl ycol ysis) were classified as heterotrophs.Similarly, OTUs encoding pathways for utilizing O 2 as a terminal electron acceptor were deemed aerobes, while those with cytoc hr omes ca pable of O 2 detoxification wer e deemed aer otoler ant.OTUs lac king homologs of O 2 -metabolizing pathw ays w ere deemed anaerobes .T he black and gray bars depict the proportions of autotrophs and heterotrophs in the community , respectively .Superimposed hashing depicts the proportions of aerotolerant and aerobic microbes, while the dots depict the proportion of anaerobes .(B) O TUs were further classified based on encoded carbon-fixing pathwa ys .Abbr e viations: WL: Wood-Ljungdahl pathwa y, rTC A: r eductiv e tricarboxylic acid cycle, and CBB: Calvin-Benson-Bassham cycle.